Synthesis, fungal biotransformation, and evaluation of the antimicrobial potential of chalcones with a chlorine atom

Chalcones are intermediate products in the biosynthesis of flavonoids, which possess a wide range of biological properties, including antimicrobial and anticancer activities. The introduction of a chlorine atom and the glucosyl moiety into their structure may increase their bioavailability, bioactivity, and pharmacological use. The combined chemical and biotechnological methods can be applied to obtain such compounds. Therefore, 2-chloro-2′-hydroxychalcone and 3-chloro-2′-hydroxychalcone were synthesized and biotransformed in cultures of two strains of filamentous fungi, i.e. Isaria fumosorosea KCH J2 and Beauveria bassiana KCH J1.5 to obtain their novel glycosylated derivatives. Pharmacokinetics, drug-likeness, and biological activity of them were predicted using cheminformatics tools. 2-Chloro-2′-hydroxychalcone, 3-chloro-2′-hydroxychalcone, their main glycosylation products, and 2′-hydrochychalcone were screened for antimicrobial activity against several microbial strains. The growth of Escherichia coli 10,536 was completely inhibited by chalcones with a chlorine atom and 3-chlorodihydrochalcone 2′-O-β-d-(4″-O-methyl)-glucopyranoside. The strain Pseudomonas aeruginosa DSM 939 was the most resistant to the action of the tested compounds. However, chalcone aglycones and glycosides with a chlorine atom almost completely inhibited the growth of bacteria Staphylococcus aureus DSM 799 and yeast Candida albicans DSM 1386. The tested compounds had different effects on lactic acid bacteria depending on the tested species. In general, chlorinated chalcones were more effective in the inhibition of the tested microbial strains than their unchlorinated counterparts and aglycones were a little more effective than their glycosides.

www.nature.com/scientificreports/viability of neutrophils and their release of reactive oxygen species than their non-chlorinated counterparts 24,27 .Similarly, the biological activity, e.g.antimicrobial activity of chalcones can be positively affected by chromatophores such as chlorine atoms.Unfortunately, there are few studies on the influence of the chlorine substituent on antimicrobial activity.In the work of Prasad et al., the chalcone derivatives substituted with halogens (chlorine and/or bromine and/or fluorine) showed significant antimicrobial activity against gram-positive bacteria Bacillus pumilus, Bacillus subtilis, and gram-negative bacteria Escherichia coli.Moreover, 4-chloro-4′-bromochalcone showed a h inhibitory effect against fungi Aspergillus niger and Rhizopus oryzae 10 .
On the other hand, the introduction of the glucose moiety, due to its hydrophilicity, positively affects the aqueous solubility of flavonoids, which is a hly important factor for its bioavailability 28,29 .In nature, most flavonoids, except catechins, are present as β-glycosides.In general, glucosides are the only glycosides that can be absorbed from the small intestine and then reach her plasma levels than compounds absorbed mostly in the colon 30,31 .Therefore, glycosylation is a common strategy to improve chemical stability, water solubility, bioavailability, and pharmacological potency of flavonoids.Enzymes called glycosyltransferases (GTs) are mainly responsible for glycosylation in living organisms and have been found in plants, animals, bacteria, and fungi [32][33][34][35][36][37] .Regarding the glycosylation of chalcones, studies using UDP-glycosyltransferase (YjiC) from Bacillus licheniformis DSM-13 have been reported.Li et al. obtained derivatives of isobavachalcone with glucosyl moiety at C-4 in ring B and C-4′ in ring A 36 .On the other hand, the reaction of YjiC with UDP-glucose and phloretin led to the formation of five different types of phloretin glucosides (with glucose moiety at C-2′, at C-4′, at C-2′, and C-4′, at C-4 and C-6′, and at C-4 and C-4′) 35 .The promising flavonoid glycosylation strategy can also be whole-cell biotransformation with entomopathogenic filamentous fungi as biocatalysts [38][39][40][41][42] .Fungal glucosyltransferases remain poorly known.However, enzymes such as UGT59A1 from Absidia caerulea and UGT58A1 from Rhizopus japonicus 43 , and glycosyltransferase-methyltransferase (GT-MT) functional module BbGT86-BbMT85 from Beauveria bassiana have been characterized 44 .Moreover, Xie et al. used a combination of genome mining and heterologous expression techniques to identify glycosyltransferase-methyltransferase (GT-MT) functional modules from other Hypocreales fungi such as Isaria fumosorosea, Claviceps purpurea, Cordyceps militaris, and Metarhizium robertsii.These GT-MT modules possess decent substrate promiscuity and regiospecificity, and can methylglucosylate flavonoid compounds, among others.
Having regard to the above, the main aim of the presented work was to obtain 2-chloro-2′-hydroxychalcone and 3-chloro-2′-hydroxychalcone and then biotransform them in cultures of entomopathogenic filamentous fungi to receive their glycosylated derivatives.We carried out microbial transformation in two strains of fungi, i.e., I. fumosorosea KCH J2 and B. bassiana KCH J1.5, resulting in the formation of 2-chlorodihydrochalcone 2′-O-β-d-(4″-O-methyl)-glucopyranoside, 3-chlorodihydrochalcone 2′-O-β-d-(4″-O-methyl)-glucopyranoside and some other glycosylated derivatives.All of these biotransformation products have not been previously described in the literature.Moreover, we employed computer-aided simulations to evaluate and compare the physicochemical properties, pharmacokinetics, and potential biological activity of all of the obtained compounds.The flavonoid aglycones (2-chloro-2′-hydroxychalcone and 3-chloro-2′-hydroxychalcone) and their glycosides obtained in the fungal biotransformation process, and also 2′-hydroxychalcone were screened for antimicrobial activity.The use of 2′-hydroxychalcone, which does not have additional substituents except 2′-hydroxyl group, made it possible to observe the influence of the presence of a chlorine atom in the structure of the tested compounds on their antimicrobial activity.
The structures of both synthesis products (3 and 5) were confirmed based on NMR (Nuclear Magnetic Resonance) spectroscopy (Tables 1 and 2 in section "Materials and methods").In the 1 H-NMR (Proton Nuclear Magnetic Resonance) spectrum of 2-chloro-2′-hydroxychalcone (3), a characteristic signal from the proton of the hydroxyl moiety at δ = 12.74 ppm with the corresponding signal from C-2′ in the 13 C-NMR (Carbon-13 Nuclear Magnetic Resonance) spectrum at δ = 164.5 ppm (Supplementary Information: Figs.S3 and S5) was observed.In the case of 3-chloro-2′-hydroxychalcone (5), this signal was observed at δ = 12.79 ppm in the 1 H-NMR spectrum with the corresponding signal at δ = 164.6 ppm in the 13 C-NMR spectrum (Supplementary Information: Figs.  and S23).In the 1 H-NMR spectra of compounds 3 and 5 were also observed characteristic two doublets from H-α (δ = 8.10 ppm in 3 and H-δ = 8.16 ppm in 5) and β (δ = 8.32 ppm in 3 and 7.91 ppm in 5) (Supplementary Information: Figs.S4 and S22).In the 13 C-NMR spectra, the signal from the carbonyl group was observed at δ = 194.8ppm in 3 and at δ = 194.9ppm in 5 (Supplementary Information: Fig. S5 and S23).The substitution with a chlorine atom in the structure of 2′-hydroxychalcone in position C-2 in ring B (product 3) confirmed the arrangement of other proton signals in ring B (from H-3 doublet of doublets at δ = 7.56 ppm, from H-4 triplet of doublets at δ = 7.50 ppm, from H-5 multiplet at δ = 7.45 ppm, and from H-6 doublet of doublets at δ = 8.17 ppm) (Supplementary Information: Fig. S4).Moreover, the signal from H-α (δ = 8.10 ppm) correlated with the shifted to the lower field signal from C-1 (δ = 133.6 ppm) in the HMBC (Heteronuclear Multiple Bond Correlation) experiment (Supplementary Information: Fig. S12).On the other hand, in the 1 H-NMR spectrum of 5, a characteristic isolated singlet from H-2 at δ = 8.01 ppm confirmed substitution with a chlorine atom in its direct neighbourhood at C-3 (Supplementary Information: Fig. S22).Moreover, the signal from H-2 (δ = 8.01 ppm) correlated with the shifted to the lower field signal from C-3 (δ = 135.4ppm) in the HMBC experiment (Supplementary Information: Fig. S30).The molecular masses of both synthesis products with chlorine atoms (3 and 5) were also confirmed by LC-MS spectroscopy (Liquid Chromatography-Mass Spectrometry) (Supplementary Information: Fig. S1 and S19, respectively).The application of entomopathogenic filamentous fungi as biocatalysts in the microbial transformation of 2-chloro-2′-hydroxychalcone (3) and 3-chloro-2′-hydroxychalcone (5) in cultures of fungi strains I. fumosorosea KCH J2 and B. bassiana KCH J1.5 resulted in the formation of six new glycosylated flavonoids.The products of the biotransformations were isolated from the post-reaction mixture and purified using preparative thin-layer chromatography (TLC).The yields of biotransformations were determined based on the isolated amounts of the products.Their chemical structures were established based on the NMR spectroscopy and confirmed by LC-MS.Their biological activity was assessed using computational methods based on the structure-activity relationship.Moreover, the antimicrobial activity of biotransformation substrates (3 and 5) and their main biotransformation products (3a, 3b, 5a) together with 2′-hydroxychalcone were screened in the experiments with the selected microbial strains using automatic measurements of their growth.
The structures of products 3a-3b were determined based on NMR spectroscopy (Tables 1 and 2 in section "Materials and methods", Figs. 4 and 5 below showing key COSY (Correlation Spectroscopy) and HMBC correlations in section "Materials and methods").Their molecular masses were confirmed using LC-MS (Section "Materials and methods" and Supplementary Information: Figs.S37 and S62).
The presence of a glucose moiety in the biotransformation product 3a was confirmed by five characteristic carbon signals observed in the region from δ = 80.1 ppm to δ = 62.1 ppm in the 13 C-NMR spectrum (Supplementary Information: Fig. S44), as well as proton signals of δ H ranging from δ = 3.83 ppm to δ = 3.20 ppm in the 1 H-NMR spectrum (Supplementary Information: Fig. S41).In the 1 H-NMR spectrum, a one-proton doublet from the proton at the anomeric carbon atom was present at δ = 5.06 ppm with the coupling constant (J = 7.8 Hz) evidencing β-configuration of the glucose (Supplementary Information: Fig. S41).The glucose molecule was also O-methylated at C-4″ because, in the 1 H-NMR spectrum, a three-proton singlet at δ = 3.55 ppm, with the corresponding signal at δ = 60.6 ppm in the 13 C-NMR spectra was observed (Supplementary Information: Figs.S41  and S44).Moreover, this moiety was correlated with the signal of C-4″ (δ = 80.1 ppm) in the HMBC experiment, which evidences the substitution position with the -O-CH 3 group in the glucose unit (Supplementary Information: Fig. S55).In product 3a, the reduction of a double bond between C-α and C-β occurred and caused the shift of the protons at C-α from δ = 8.10 ppm (in 3) to δ = 3.46 ppm (in 3a) and the protons at C-β from δ = 8.32 ppm (in 3) to δ = 3.10 ppm (in 3a) (Supplementary Information: Figs.S4 and S41).Naturally, shifted protons at C-α and C-β were correlated with the C-1 signal and the carbonyl group in the HMBC experiment (Supplementary Information: Figs.S54 and S56).The signal from one proton of the hydroxyl group at C-2′ that was present in the 1 H-NMR spectrum of the biotransformation substrate 3 at δ = 12.74 ppm was absent in the 1 H-NMR of the biotransformation product 3a (Supplementary Information: Fig. S3 and S39), which pointed at substitution at In product 3b the 4″-O-methylglucosyl moiety was also attached to the flavonoid aglycone in the β-configuration (Supplementary Information: Figs.S66 and S70).In this biotransformation product also occurred the reduction of a double bond between C-α and C-β (Supplementary Information: Figs.S66 and S70).The Table 1. 1 H-NMR chemical shifts δ (ppm) and coupling constants J (Hz) of 2-chloro-2′-hydroxychalcone (3) and products of its biotransformation 3a-3c in Acetone-d6, 600 MHz (Supplementary Information: Figs.S3-S4, S39-S41, S64-S66, S90-S92).www.nature.com/scientificreports/3b) (Supplementary Information: Figs.S4 and S65).The absence of the signal from H-5′ in the neighborhood of H-3′ (visible in substrate 3) was also evident, which indicated substitution with the glucosyl moiety at C-5′ (Supplementary Information: Figs.S4 and S65).In the HMBC experiment, the signal from H-1″ at δ = 4.85 ppm correlated with the shifted signal at δ = 150.8ppm, which was assigned as C-5′ (Supplementary Information: Fig. S80).Moreover, the signal at δ = 150.8ppm correlated with H-6′ at δ = 7.66 ppm, H-4′ at δ = 7.30 ppm, and H-3′ at δ = 6.88 ppm, which proved that it came from C-5′ (Supplementary Information: Fig. S79).
This biotransformation product was a result of a few chemical reactions resulting in the glycosylated and hydroxylated derivative.Firstly, compound 3c was identified as dihydrochalcone because the reduction of a double bond between C-α and C-β occurred causing chemical shifts in the location of the signal of H-α (from δ = 8.10 ppm in 3 to δ = 3.44 ppm in 3c) and the signal of H-β (from δ = 8.32 ppm in 3 to δ = 3.14 ppm in 3c) in the 1 H-NMR spectrum (Supplementary Information: Figs.S4 and S92).Secondly, the signal from one proton of the hydroxyl group at C-2′ was shifted from δ = 12.74 ppm (in biotransformation substrate 3) to δ = 12.07 ppm (in biotransformation product 3c), which indicated the substitution in ring A of the product 3c (Supplementary www.nature.com/scientificreports/Information: Figs.S3 and S90).This substituent was a glucose moiety because in the 1 H-NMR and 13 C-NMR spectra were observed characteristic signals, similar to the above mentioned for product 3a (Supplementary Information: Figs.S92 and S95).It was attached at C-3′ because it's signal (δ = 147.2ppm), which correlated with H-1″ from the glucosyl moiety (δ = 4.91 ppm) in the HMBC experiment, correlated also with 2′-OH (δ = 12.07 ppm), H-4′ (δ = 7.40 ppm), and H-5′ (δ = 6.89 ppm) (Supplementary Information: Figs.S106, S103, and S104).Thirdly, another signal from the proton of the hydroxyl group at δ = 8.70 ppm was observed.Along with the observed shifts in the position of protons coming from the B ring of product 3c, it indicated substitution in ring B. The hydroxyl group was attached at C-3 which resulted in the shift of H-5 (from δ = 7.45 ppm in 3 to δ = 7.09 ppm in 3c) H-4 (from δ = 7.50 ppm in 3 to δ = 6.89 ppm in 3c) and H-6 (from δ = 8.17 ppm in 3 to δ = 6.89 ppm in 3c) (Supplementary Information: Figs.S90 and S91).Moreover, in the HMBC experiment, the proton of the hydroxyl group at δ = 8.70 ppm (assigned as 3-OH) correlated with C-4 (δ = 115.4ppm) and C-6 (δ = 122.1 ppm) (Supplementary Information: Fig. S104).Furthermore, protons H-5 (δ = 7.09 ppm) and H-4 (δ = 6.89 ppm) correlated also with C-3 (δ = 154.2ppm) (Supplementary Information: Fig. S104).
The structures of products 5a-5b were determined based on NMR spectroscopy (Tables 3 and 4 in section "Materials and methods", Figs. 9 and 10 below showing key COSY and HMBC correlations).Their molecular masses were confirmed using LC-MS (section "Materials and methods" and Supplementary Information: Figs.S113 and S138).
The product 5a was analogous to product 3a (the only difference between them is the position of substitution with the chlorine atom).In this case also occurred the attachment of 4″-O-methylglucosyl moiety in β-configuration at C-2′ because in the 1 H-NMR spectrum, the signal from 2′-OH disappeared and characteristic signals from the glucose moiety were observed (Supplementary Information: Figs.S115, S117, and S120).Moreover, in 5a, similarly to 3a, there was a reduction of a double bond between C-α and C-β, causing characteristic chemical shifts in the 1 H-NMR spectrum (Supplementary Information: Figs.S22, S24, S117, and 120).
The compound 5b was analogous to product 3b (the only difference between them is the position of substitution with the chlorine atom).In this biotransformation product, the 4″-O-methylglucosyl moiety was attached to the flavonoid aglycone in the β-configuration (Supplementary Information: Figs.S142 and S145).The reduction of a double bond between C-α and C-β was confirmed by 1 H-NMR and 13 C-NMR spectra (Supplementary Information: Figs.S142 and S145).The signal from one proton of the hydroxyl group at C-2′ in the 1 H-NMR spectrum was shifted from δ = 12.79 ppm (in biotransformation substrate 5) to δ = 11.88 ppm (in biotransformation product 5b), which indicated that the substitution with the glucosyl moiety occurred in ring A of the flavonoid compound (Supplementary Information: Figs.S21 and S140).Similarly, to 3b, the absence of the signal from H-5′ in the neighborhood of H-3′ (visible in substrate 5) indicated substitution with the glucosyl moiety at C-5′ (Supplementary Information: Figs.S22 and S141).Furthermore, in the HMBC experiment, the signals from H-6′ at δ = 7.67 ppm, H-4′ at δ = 7.29 ppm, H-3′ at δ = 6.87 ppm, and H-1″ at δ = 4.85 ppm correlated with the shifted signal at δ = 150.8ppm (assigned as C-5′), which proved substitution at C-5′ (Supplementary Information: Figs.S155 and S156).

Biotransformation of 3-chloro-2′-hydroxychalcone (5) in the culture of B. bassiana KCH J1.5
Biotransformation of 3-chloro-2′-hydroxychalcone (5) in the culture of B. bassiana KCH J1.5 resulted in the formation of three products, i.e., 3-chlorodihydrochalcone 2′-O-β-d-(4″-O-methyl)-glucopyranoside (5a) with a yield of 14.7% (12.4 mg), 3-chloro-2′-hydroxydihydrochalcone 5′-O-β-d-(4″-O-methyl)-glucopyranoside (5b) with a yield of 10.3% (9.0 mg), and 3-chloro-2′-hydroxydihydrochalcone 4-O-β-d-(4″-O-methyl)glucopyranoside (5c) with a yield of 6.6% (5.8 mg) (Fig. 11).The first two listed, based on the 1 H-NMR analysis, were identified as the same products (5a and 5b) as obtained in the biotransformation in the culture of I. fumosorosea KCH J2.The structure of the third, unknown product 5c was determined based on NMR spectroscopy (Tables 3 and 4 in section "Materials and methods", Fig. 12 below showing key COSY and HMBC correlations).Its molecular mass was confirmed using LC-MS (section "Materials and methods" and Supplementary Information: Fig. S163).www.nature.com/scientificreports/ The last biotransformation product 5c was glycosylated because the characteristic signals of 4″-O-methylglucosyl moiety were present in the 1 H-NMR and 13 C-NMR spectra (Supplementary Information: Figs.S167  and S170).In this product occurred also the reduction of a double bond between C-α and C-β (Supplementary Information: Figs.S167 and S170).The signal from one proton of the hydroxyl group at C-2′, similar to the other signals of protons of ring A, was only slightly shifted, compared to substrate 5, because of mentioned hydrogenation of the double bond between C-α and C-β (Supplementary Information: Figs.S21, S22, S165, and 166).The substitution with the glucosyl moiety occurred in ring B at C-4.The signal from the H-2 proton in the 1 H NMR spectrum of product 5c is a doublet with a coupling constant of J = 1.9, which indicates that only one meta hydrogen (H-6) is in its vicinity.Such an arrangement is possible if the glycosidic unit was attached to the C-4.The 1 H-NMR spectrum also shows a doublet (δ = 7.30) with a coupling constant of J = 8.1, which indicates that there is a proton in the ortho position in relation to it (H-5).The COSY spectrum also confirms the position of the substitution by the presence of coupling of the analyzed signal with the signal originating from the H-6 proton (Supplementary Information: Fig. S180).
Both biotransformation substrates, i.e. 2-chloro-2′-hydroxychalcone (3) and 3-chloro-2′-hydroxychalcone (5) were biotransformed in cultures of fungi strains I. fumosorosea KCH J2 and B. bassiana KCH J1.5 into glycosylated dihydrochalcones.The main products were obtained by the attachment of 4″-O-methylglucosyl moiety to the hydroxyl group at C-2′ in the A ring.In our previous works on methylchalcones 42,45 , products of Table 3. 1 H-NMR chemical shifts δ (ppm) and coupling constants J (Hz) of 3-chloro-2′-hydroxychalcone (5)  and products of its biotransformation 5a-5c in Acetone-d6, 600 MHz (Supplementary Information: Figs..However, effective glycosylation of chlorochalcones was previously described only by our team 49 .Unfortunately, it is difficult to draw a universal conclusion on the impact of glycosylation on the biological activity of flavonoids.Some studies showed a reduction of.e.g.anti-oxidant, anti-inflammatory, antibacterial activity, and others described an increase in, e.g.tyrosinase inhibition, antivirus, and antiallergic activity.The effects of flavonoid glycosylation may likely differ in in vitro and in vivo studies 29 .

Antimicrobial activity of 2-chloro-2′-hydroxychalcone (3), 3-chloro-2′-hydroxychalcone (5), 2′-hydroxychalcone (6) and their selected derivatives (3a and 5a)
Predictions have not shown with h confidence target microorganisms of antimicrobial activity of the obtained compounds but pointed out that they may be effective in inhibition of CDP-glycerol glycerophosphotransferase, which is an important enzyme in the pathogenesis of Gram-positive bacteria.Therefore, we decided to make screening tests of the antimicrobial activity of 2-chloro-2′-hydroxychalcone (3), 3-chloro-2′-hydroxychalcone (5), their main products of fungal biotransformation products, i.e., 2-chlorodihydrochalcone 2′-O-β-d-(4″-Omethyl)-glucopyranoside (3a) and 3-chlorodihydrochalcone 2′-O-β-d-(4″-O-methyl)-glucopyranoside (3a), and for comparision 2′-hydroxychalcone (6) using the Bioscreen C (Automated Microbiology Growth Curve Analysis System) against three strains of bacteria Escherichia coli 10,536 (Gram-), Pseudomonas aeruginosa DSM 939(Gram-), Staphylococcus aureus DSM 799 (Gram +), one strain of yeast Candida albicans DSM 1386, and three strains of lactic acid bacteria Lactococcus acidophilus KBiMZ 01 (Gram +), Lactococcus rhamnosus GG (Gram +), Streptococcus thermophilus KBM-1 (Gram +).The obtained data on the duration of the lag-phase in microbial cultures, both control (only with the tested strain) and with the addition of the tested compounds, as well as the biomass increase, expressed as an increase in optical density (ΔOD), are shown in Tables 8 and 9. Table 6.Predictions of biological activities and targets of compounds 3, 3a-3c, and 6 using Way2Drug Pass Online tool.a Pa-probable activity.b Pi-probable inactivity.Values range from 0 to 1, where 1 represents a 100% probability of Pa or Pi, and 0 represents a 0% probability of Pa or Pi.c JAK2-Janus kinase 2-widely expressed tyrosine kinase responsible for signal transduction, plays a significant role in hematopoiesis, a therapeutic target as its mutations are related to malignant transformations 55 .d CDPglycerol glycerophosphotransferase-an enzyme responsible for the polymerization of teichoic acid chains, which plays a key role in shaping the bacterial cell, integrating its envelope, creating a bacterial biofilm, and, consequently, the pathogenesis of gram-positive bacteria 56 .e Anaphylatoxin-complement peptide, which plays a role in response to bacterial infections and inflammatory processes, sepsis, ischemia-reperfusion injuries, complex immunological diseases, and asthma 57 .f Monophenol monooxygenase-the rate-limiting enzyme for controlling the production of melanin, its increased activity is associated with the development of malignant melanoma 58 .g G-protein-coupled receptor kinases-family of enzymes that regulate the function of G-proteincoupled receptors that are cellular sensors involved in many physiological processes; increased activity of these kinases contributes to the loss of contractile reserve in the stressed and failing heart, therefore their inhibition is one of the new therapeutic approaches in the treatment of heart failure 59 .Biological assays performed on the obtained flavonoids allowed us to evaluate the effect the of introduction of a chlorine atom and 4′-O-methylglucosyl moiety into flavonoid structure on antimicrobial activity.The strongest inhibitory effect against E. coli 10,536 (Gram -) was observed for flavonoid aglycones 3 (ΔOD = 0) and 5 (ΔOD = 0.06) as well as for glycoside 5a (ΔOD = 0.06).Other compounds also significantly inhibited bacterial growth, compound 6 with ΔOD = 0.25 and 3a with ΔOD = 0.26.The growth of the E. coli strain against the tested compounds is shown in Fig. 13.
The tested strain of another Gram-bacteria, i.e., P. aeruginosa DSM 939 was more resistant to the action of the tested compounds and its growth was most strongly inhibited only by compound 5 (ΔOD = 0.11).All other compounds showed significant inhibition of the growth of these bacteria, however, the increase in optical Table 7. Predictions of biological activities and targets of compounds 5, 5a-5c, and 6 using Way2Drug Pass Online tool.a Pa-probable activity.b Pi-probable inactivity.Values range from 0 to 1, where 1 represents a 100% probability of Pa or Pi, and 0 represents a 0% probability of Pa or Pi.c JAK2-Janus kinase 2-widely expressed tyrosine kinase responsible for signal transduction, plays a significant role in hematopoiesis, a therapeutic target as its mutations are related to malignant transformations 55 .d CDP-glycerol glycerophosphotransferase-an enzyme responsible for the polymerization of teichoic acid chains, which plays a key role in shaping the bacterial cell, integrating its envelope, creating a bacterial biofilm, and, consequently, the pathogenesis of gram-positive bacteria 56 .e Anaphylatoxin-complement peptide, which plays a role in response to bacterial infections and inflammatory processes, sepsis, ischemia-reperfusion injuries, complex immunological diseases, and asthma 57 .f Monophenol monooxygenase-the rate-limiting enzyme for controlling the production of melanin, its increased activity is associated with the development of malignant melanoma 58 .g G-protein-coupled receptor kinases-family of enzymes that regulate the function of G-proteincoupled receptors that are cellular sensors involved in many physiological processes; increased activity of these kinases contributes to the loss of contractile reserve in the stressed and failing heart, therefore their inhibition is one of the new therapeutic approaches in the treatment of heart failure 59 .www.nature.com/scientificreports/density was not as low as in the case of E. coli and ranged from 0.22 to 0.36 (3 -ΔOD = 0.22, 3a -ΔOD = 0.24, 5a -ΔOD = 0.35, and 6 -ΔOD = 0.36), while the value of this parameter for the control was 0.63.The growth of the P. aeruginosa strain against the tested compounds is shown in Fig. 14.
The tested compounds at the concentration of 0.1% had different effects on lactic acid bacteria (Gram +) depending on the tested species.The growth of L. acidophilus KBiMZ 01 bacteria was completely inhibited only by the compounds 5, 5a, and 6.Compounds 3 and 3a limited the growth of the tested bacterial strain causing the OD to increase to 0,30 and 0,36, respectively, while the value of this parameter for the control was 1.73.The strains L. rhamnosous GG and S. thermophilus KBM-1 were very sensitive to the action of the tested compounds.In the case of L. rhamnosus PAM, a slight increase in OD, at the level of 0.22, was observed only in the case of compound 5a.The effect of the action of the tested compounds on lactic acid bacteria is shown in Figs.17, 18, and 19.
The screening of antimicrobial activity of 2-chloro-2′-hydroxychalcone (3), 3-chloro-2′-hydroxychalcone ( 5), their 2′-O-β-d-(4″-O-methyl)-glucopyranosides (3a and 5a) and 2′-hydroxychalcone (6) revealed that an introduction of a chlorine atom into the flavonoid structure increased their inhibitory effect againt E. coli 10,536, S. aureus DSM 799, P. aeruginosa DSM 939, and C. albicans DSM 1386.A similar effect was observed in the work of Prasad et al., in which chalcones with pharmacophores such as chloro, bromo, and fluoro groups exhibited h antimicrobial and antifungal potential 10 .Likewise, the introduction of a chlorine or bromine atom positively affected the action of pyrazine-based chalcones, which showed anti-staphylococcal and anti-enterococcal activity 60 .In the evaluation of chalcones' inhibitory activity against Mycobacterium tuberculosis H37Rv, the substitution of a halogen atom on ring B of 2'-hydroxychalcone increased its anti-tuberculosis activity.Compounds with a halogen at C-3 demonstrated stronger anti-tuberculosis activity than those with a halogen substituent at C-2 or C-4.3-Chloro-2′-hydroxychalcone exhibited a h 90% inhbition againt M. tuberculosis H37RV at a concentration 12.5 mg/mL 61 .These results are consistent with our findings.In the evaluation of the antibacterial activity of flavonoid glycosides (flavonol 3-O-glycosides), strong inhibition against Gram-positive bacteria was observed.In addition, Gram-positive bacteria were more sensitive than the Gram-negative bacteria 29 .The results of our studies weren't so consistent because Gram-negative bacteria E. coli 10,536 was also hly sensitive to the action of both flavonoid aglycones and glycosides.Certainly, there is a need for further, more detailed assessment of the antimicrobial activity of the obtained compounds, including learning their mechanisms of action, the effect of the chlorine and glucosyl substituent, and conducting in vivo studies as well.

Conclusions
Functionalization of chalcones can be achieved by introducing a chlorine atom and glucosyl moiety into their structure, which leads to the compounds with increased water solubility and altered biological activity.In the presented paper, we described the great capacity of entomopathogenic fungi strains I. fumosorosea KCH J2 and B. bassiana KCH J1.5 to produce dihydrochalcone 4-O-methylglucosides from synthetic chalcones, i.e. 2-chloro-2′hydroxychalcone and 3-chloro-2′-hydroxychalcone.In both biotransformations, the main glycosylated products were obtained with good yields (3a-74.5% and 3b-41% in the case of using culture of I. fumosorosea KCH www.nature.com/scientificreports/J2) by the attachment of 4″-O-methylglucosyl moiety to the hydroxyl group at C-2′ in ring A and the reduction of the double bond.All six biotransformation products have not been described in the literature until now.The methods described in this paper allow the effective and relatively cheap preparation of large amounts of glycoside derivatives with a chlorine atom for further studies of their biological activity and bioavailability.We performed computational studies of newly prepared compounds based on the structure-activity relations to preliminary assess their biological activities and accelerate screening procedures for further research.However, in vitro and in vivo biological studies are still necessary for a full understanding of the biological activities, pharmacokinetics, and molecular mechanisms of action of potential new drugs.Our screening of the antimicrobial activity of the obtained compounds showed that the introduction of a chlorine atom in the structure of 2′-hydroxychalcone increases antimicrobial activity against tested strains, but we didn't observe increased activity of flavonoid glycosides compared to aglycones against Gram-positive bacteria, as suggested by the results of Pass Online simulations.Further research into biological activity, such as membrane integrity agonist, antimicrobial, antiprotozoal, anticancer, anti-inflammatory activity, and metabolic stability, is needed both to assess the usefulness of newly obtained compounds in medicine and to better train algorithms used in chemistry-informatics tools for predicting the biological potential of chemical compounds.
The physical data, including color and form, molecular ion mass, molecular formula, melting point (°C), retention time t R (min), retardation factor Rf, and NMR spectral data of the flavonoids 3 and 5 are presented below, in Tables 1, 2, 3, and 4, and the Supplementary Information.

Microorganisms
Biotransformation of chlorochalcones 3 and 5 was performed using two entomopathogenic filamentous fungi strains belonging to the family Cordycipitaceae, i.e., I. fumosorosea KCH J2 and B. bassiana KCH J1.5.Both strains came from the collection of the Faculty of Biotechnology and Food Microbiology of the Wrocław University of Environmental and Life Sciences in Poland.A detailed description of the collection and reproduction of these fungi as well as their genetic identification were described in our previous works 63,64 .Microbes used in antimicrobial activity screening tests, i.e., E. coli 10,536 (Gram-negative), P. aeruginosa DSM 939 (Gram-negative), S. aureus DSM 799 (Gram-positive), three strains of Gram-positive lactic bacteria S. thermophilus KBM-1, L. acidophilus KBiMZ 01, and L. rhamnosus GG, and also yeast strain C. albicans DSM 1386 also belong to the collection of the Faculty of Biotechnology and Food Microbiology of Wrocław University of Environmental and Life Sciences.

Analysis
Thin-layer chromatography (TLC) and h-performance liquid chromatography (HPLC) were used to assess the course of biotransformation, in particular substrate conversion.
HPLC analyses were performed using a Dionex Ultimate 3000 instrument (Thermo Fisher Scientific, Waltham, MA, USA) with a DAD-3000 diode array detector and analytical octadecyl silica (ODS) 2 column (4.6 mm × 250 mm, Waters, Milford, MA, USA) and dedicated pre-column.The mobile phase was a mixture of 0.1% formic acid (Honeywall, Charlotte, North Carolina, US), in water (Supelco, Darmstadt, Germany) v/v (A) and 0.1% formic acid in acetonitrile (Supelco, Darmstadt, Germany) v/v (B).The gradient program was as follows: initial conditions-32.5% B in A, 4 min-40% B in A, 8 min-40% B in A, 10 min-45% B in A, 15 min-95% B in A, 18 min-95% B in A, 19 min-32.5% B in A, 23 min-32.5% B in A. The flow rate was 1 mL/min, the injection volume was 10 µL of samples in a concentration of 1 mg/ml (dissolved in acetonitrile), and the detection wavelengths were 254 (flavonoid glycosides) and 280 nm (flavonoid aglycones) 42 .All compounds are > 95% pure by HPLC analysis.
The separation of the biotransformation products obtained on the semipreparative scale was attained with the use of 500 µm and 1000 µm preparative TLC silica gel plates (Analtech, Gehrden, Germany) with a mixture of chloroform and methanol (9:1 v/v) as eluent.The compounds were extracted from the selected gel fractions www.nature.com/scientificreports/

Pharmacokinetics, drug nature, biological activity predictions
The predictions of pharmacokinetics, physicochemical properties, drug nature, and potential biological activities of flavonoids 3, 3a-3c, 5, 5a-5c, 6 based on their structural formulae were computed using the SwissADME (available online: http:// www.swiss adme.ch (accessed on October 10 th ) and Way2Drug Pass Online with accompanying services (available online: http:// www.way2d rug.com/ PASSO nline, https:// www.way2d rug.com/ antib ac, https:// www.way2d rug.com/ micF, https:// www.way2d rug.com/ antiv ir (accessed on October 10th).The structures of the molecules were built by ACD Chemsketch 2021.2.0 and saved in a .molformat and, in this form imported into both online tools.The biological activity types in Pass Online are shown as the probability to be revealed (Pa) and not to be revealed (Pi) and are independent values in the range from 0 to 1.

Antimicrobial activity assays
The study of the antimicrobial activity of compounds 3, 3a, 3b, 5, 5a, and 6 (purchased from Sigma-Aldrich, Sant Louis, MO, USA) was performed using the Bioscreen C (Automated Microbiology Growth Curve Analysis System, Helsinki, Finland) against the following strains of bacteria: E. coli 10,536 (Gram-negative), P. aeruginosa DSM 939 (Gram-negative), S. aureus DSM 799 (Gram-positive), three strains of Gram-positive lactic bacteria S. thermophilus KBM-1, L. acidophilus KBiMZ 01, and L. rhamnosus GG, and also yeast strain C. albicans DSM 1386.All the microorganisms belong to the collection of the Faculty of Biotechnology and Food Microbiology of Wrocław University of Environmental and Life Sciences.The microbiological cultures were cultured before assays for 48 h: bacteria E. coli 10,536, P. aeruginosa DSM 939, S. aureus DSM 799 in a liquid broth LB (5 g of yeast extract, 10 g of tryptone, and 10 g of NaCl, dissolved in 1 L of distilled water (purchased from Merck, Darmstadt, Germany), yeasts C. albicans DSM 1386 in liquid YPG medium (10 g of yeast extract, 10 g of bacteriological peptone, and 10 g of glucose, dissolved in 1 L of distilled water, bacteria S. thermophilus KBM-1 in M17 medium (0.5 g of ascorbic acid, 5 g of lactose, 0.25 g of magnesium sulfate, 5 g of meat extract, 2.5 g of meat peptone, 19 g of sodium glycerophosphate, 5 g of soya peptone, 2.5 of tryptone, and 2.5 g of yeast extract, dissolved in 1 L of distilled water, bacteria L. acidophilus KBiMZ 01 and L. rhamnosus GG in liquid MRS medium (4 g of yeast extract, 2 g of triammonium citrate, 5 g of sodium acetate trihydrate, 10 g of sodium acetate trihydrate, 10 g of peptone, 8 g of meat extract, 0.05 g of manganous sulfate tetrahydrate, 0.2 g of magnesium sulfate heptahydrate, 20 g of glucose, 2 g of dipotassium hydrogen phosphate, dissolved in 1 L of distilled water.Tests were performed on 100-well microtiter Bioscreen C plates, with the working volume in each well of 300 μL, comprising 280 μL of culture medium, and 10 μL of microorganism suspension with the final density of 1x10 6 cells/mL, and tested flavonoids 10 μL (dissolved in dimethyl sulfoxide, final concentration 0.1% (m/v)).The temperature was controlled at 30˚C and the optical density was measured automatically 72 h at regular intervals of 60 min at 560 nm in the case of E. coli 10,536, P. aeruginosa DSM 939, S. aureus DSM 799, C. albicans DSM 1386 and at 37˚C and the optical density was measured automatically 70 h at regular intervals of 30 min at 560 nm in the case of lactic acid bacteria.Each culture was prepared in three replications and continuously shaken during the experiment.Oxytetracycline-a broad-spectrum oxytetracycline antibiotic (10 mg/ml; Sigma-Aldrich, Saint Louis, MO, USA) and cycloheximide-a naturally occurring fungicide (0,1% (m/v); Sigma-Aldrich, Saint Louis, MO, USA) were used as positive controls.The obtained data was analyzed using Microsoft Excel software.To prepare the growth curves for each strain, the mean values of the absorbance of the medium as a function of time were used.The resulting antimicrobial activity was expressed as the increase in optical density (ΔOD) and was compared to that of the control cultures in the medium with only dimethyl sulfoxide added. S21

Figure 5 .Figure 6 .Figure 7 .
Figure 5. Key COSY (on the left) and HMBC (on the right) correlations for the structure elucidation of product 3b.

Figure 9 .Figure 10 .
Figure 9. Key COSY (on the left) and HMBC (on the right) correlations for the structure elucidation of product 5a.

Figure 12 .
Figure 12.Key COSY (on the left) and HMBC (on the right) correlations for the structure elucidation of product 5c.

Figure 16 .
Figure 16.The effect of the action of compounds 3, 5, their main biotransformation products (3a, 5a), and 6 on the growth of C. albicans DSM 1386.

Table 5 .
Pharmacokinetics, drug-likeness, and biological activity prediction data from the SwissADME online tool of compounds 3